Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT’s developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.

NOTCH1 is a transmembrane receptor in metazoans that is linked to a variety of disorders. The receptor contains an extracellular domain (ECD) with 36 tandem epidermal growth factor-like (EGF) repeats. The ECD is responsible for intercellular signaling via protein–ligand interactions with neighboring cells. Each EGF repeat consists of approximately 40 amino acids and 3 conserved disulfide bonds. The Abruptex region (EGF24-29) is critical for NOTCH1 signaling and is known for its missense mutations. Certain EGF repeats are modified with the addition of O-linked glycans and many have calcium binding sites, which give each EGF repeat a unique function. It has been shown that the loss of the O-fucose site of EGF27 alters NOTCH1 activity. To investigate the role of glycosylation in the NOTCH1 signaling pathway, nuclear magnetic resonance spectroscopy has been employed to study the structures of EGF27 and its glycoforms. Here, we report the backbone and sidechain 1H, 15N, and 13C-resonance assignments of the unmodified EGF27 protein and the predicted secondary structure derived from the assigned chemical shifts.

Chlamydia trachomatis is an obligate intracellular bacterium that causes the most common sexually transmitted bacterial diseases in the world. With a biphasic developmental cycle, the bacteria utilize a type III secretion system (T3SS) to invade host cells as infectious elemental bodies, which then differentiate into actively dividing reticulate bodies. The regulation of the developmental cycle and the T3SS are linked by the bi-functional protein, specific Chlamydia chaperone 4 (Scc4). Scc4 is a class I T3SS chaperone forming a heterodimer with specific Chlamydia chaperone 1 (Scc1) to chaperone the essential virulence effector, Chlamydia outer membrane protein N. Scc4 also functions as a transcription factor by binding to the RNA polymerase holoenzyme between the flap region of the β subunit and region 4 of σ66. In order to investigate the mechanism behind Scc4’s dual functions and target its protein-protein interactions as a route for drug development, the structure and dynamics of Scc4 are being pursued. In the course of this effort, we assigned 89.2% of the backbone and sidechain 1H, 15N, and 13C resonances of full-length Scc4. The assigned chemical shifts were used to predict the secondary structure and dynamic properties. The type and order of Scc4’s determined secondary structure are consistent with the X-ray crystal structures of other bacterial T3SS chaperones.

Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4's contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4's effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the β-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ70) (homologous to chlamydial σ66) mutants that strengthen the interaction between σ70 region 4 and the β-flap, confirming Scc4's distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS.IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).

Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the 15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4′s functional switching mechanism and Scc4:Scc1 association in CT.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes the most common sexually transmitted bacterial disease in the world. The bacterium has a unique biphasic developmental cycle with a type III secretion system (T3SS) to invade host cells. Scc4 is a class I T3SS chaperone forming a heterodimer complex with Scc1 to chaperone the essential virulence effector, CopN. Scc4 also functions as an RNA polymerase binding protein to regulate σ66-dependent transcription. Aggregation and low solubility of 6X-histidine-tagged Scc4 and the insolubility of 6X-histidine and FLAG-tagged Scc1 expressed in Escherichia coli have hindered the high-resolution nuclear magnetic resonance (NMR) structure determination of these proteins and motivated the development of an on-column complex dissociation method to produce tag-free Scc4 and soluble FLAG-tagged Scc1. By utilizing a 6X-histidine-tag on one protein, the coexpressed Scc4-Scc1 complex was captured on nickel-charged immobilized metal affinity chromatography resin, and the nondenaturing detergent, sodium N-lauroylsarcosine (sarkosyl), was used to dissociate and elute the non-6X-histidine-tagged protein. Tag-free Scc4 was produced in a higher yield and had better NMR spectral characteristics compared to 6X-histidine-tagged Scc4, and soluble FLAG-tagged Scc1 was purified for the first time in a high yield. The backbone structure of Scc4 after exposure to sarkosyl was validated using NMR spectroscopy, demonstrating the usefulness of the method to produce proteins for structural and functional studies. The sarkosyl-assisted on-column complex dissociation method is generally applicable to protein complexes with high affinity and is particularly useful when affinity tags alter the protein's biophysical properties or when coexpression is necessary for solubility.

Peptides that exhibit enzymatic or hormonal activities are regulatory factors and desirable therapeutic drugs because of their high target specificity and minimal side effects. Unfortunately, these drugs are susceptible to enzymatic degradation, leading to their rapid elimination and thereby demanding frequent dosage. Structurally modified forms of some peptide drugs have shown enhanced pharmacokinetics, improving their oral bioavailability. Here, we discuss a novel glycomimetic approach to modify lysine residues in peptides. In a model system, the ε-amine of Ts-Lys-OMe was reductively alkylated with a glucose derivative to afford a dihydroxylated piperidine in place of the amine. A similar modification was applied to H-KPV-NH2, a tripeptide derived from the α-melanocyte stimulating hormone (α-MSH) reported to have antimicrobial and anti-inflammatory properties. Antimicrobial assays, under a variety of conditions, showed no activity for Ac-KPV-NH2 or the α- or ε-glycoalkylated analogs. Glycoalkylated peptides did, however, show stability toward proteolytic enzymes.

Polyhydroxyalkanoates (PHAs) are gaining popularity as a viable biological alternative to petroleum-derived polyolefin plastic materials, especially because their material properties can be tuned through monomer composition and configuration, an uncommon phenomena in enzymatic polymer products. Because the native function of these polymers is chemical energy storage, the metabolic synthesis routes can vary dramatically from one microorganism to the next, save one crucial enzyme - the PHA synthase, responsible for the direct polymerization and composition of PHAs. For this reason, the field of PHA synthase mechanics is blooming as more variants of this enzyme are discovered. However, due to the complexity associated with this surface-driven micelle growth reaction, the initial reaction conditions dictate a variety of observed behaviors during in vitro kinetic analysis. In this work, we discuss the importance of these initial conditions and their associated behaviors and propose minimal kinetic models to help elucidate the significant mechanics of this complex in vitro reaction. The theory and inferences of this work provide a more complete understanding of in vitro PHA synthesis and aid in the design of future in vitro PHA synthase enzymatic assays. (C) 2016 Elsevier Ltd. All rights reserved.

Background: The type III secretion (T3S) chaperone Scc4 modulates Chlamydia RNA polymerase holoenzyme activity and is also required for secretion of the gatekeeper CopN. Results: Interactions between the Scc4 and Scc1 chaperones and CopN are characterized. Conclusion: Scc4 forms a ternary complex with Scc1 and CopN to promote CopN secretion during infection. Significance: Scc4 is an important link between the T3S system and transcription. Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818–1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.